Cardiac Macrophage Infiltration Research Articles

Exercise Training Attenuates Acute β-Adrenergic Receptor Activation-Induced Cardiac Inflammation via the Activation of AMP-Activated Protein Kinase.

Exercise has proven cardiac benefits, but the underlying mechanisms of exercise that protect the heart from acute sympathetic stress injuries remain unknown. In this study, adult C57BL/6J mice and their AMP-activated protein kinase α2 knockout (AMPKα2-/-) littermates were either subjected to 6 weeks of exercise training or housed under sedentary conditions and then treated with or without a single subcutaneous injection of the β-adrenergic receptor (β-AR) agonist isoprenaline (ISO). We investigated the differences in the protective effects of exercise training on ISO-induced cardiac inflammation in wild-type (WT) and AMPKα2-/- mice using histology, enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. The results indicated that exercise training alleviated ISO-induced cardiac macrophage infiltration, chemokines and the expression of proinflammatory cytokines in wild-type mice. A mechanism study showed that exercise training attenuated the ISO-induced production of reactive oxygen species (ROS) and the activation of NLR Family, pyrin domain-containing 3 (NLRP3) inflammasomes. In cardiomyocytes, the ISO-induced effects on these processes were inhibited by AMP-activated protein kinase (AMPK) activator (metformin) pretreatment and reversed by the AMPK inhibitor (compound C). AMPKα2-/- mice showed more extensive cardiac inflammation following ISO exposure than their wild-type littermates. These results indicated that exercise training could attenuate ISO-induced cardiac inflammation by inhibiting the ROS-NLRP3 inflammasome pathway in an AMPK-dependent manner. Our findings suggested the identification of a novel mechanism for the cardioprotective effects of exercise.

Characteristics of the Cardiosplenic Axis in Patients with Fatal Myocardial Infarction.

Myocardial ischemia triggers neurohumoral activation of the cardiosplenic axis. In rodents, adverse outcomes occur upon prolonged entrance of mononuclear cells from the spleen into myocardial tissue. The purpose of this study is to assess the features of spleen structure in patients with fatal myocardial infarction (MI), the dynamics of macrophage infiltration of the spleen and its relationship with cardiac macrophage infiltration and unfavorable outcomes. Using immunohistochemistry techniques, we analyzed the macrophage infiltration of the spleen and myocardium sections collected from patients (n = 30) with fatal MI. The spleen of the patients was decreased and showed a predominance of red pulp with a high concentration of CD68+ and stabilin-1+ cells. The white pulp contained many medium and small follicles and a lower concentration of CD68+ and stabilin-1+ cells, which was comparable to that in the infarct area of the myocardium. The concentration of CD68+ and stabilin-1+ cells increased in the myocardium in the late period of MI, but did not show any dynamics in the spleen. A high number of CD68+ cells in the red pulp and reduced concentration of stabilin-1+ cells in the white pulp were associated with unfavorable post-infarction outcomes. These fundamental findings could be a basis for the development of new personalized therapeutic and diagnostic approaches for the treatment of MI and its complications.

Macrophage depletion protects against endothelial dysfunction and cardiac remodeling in angiotensin II hypertensive mice

ABSTRACT Objective: Hypertension is associated with a low-grade systemic inflammation in cardiovascular system. Macrophage infiltration may initiate an inflammatory process that contributes to vascular and ventricular remodeling in hypertensive human and mice. The present study investigated the effect of chemical depletion of macrophage using liposome encapsulated clodronate (LEC) on cardiac hypertrophy and remodeling in angiotensin (Ang) II hypertensive mice. Methods: C57BL/6 mice received an Ang II (1.1 mg/kg/day with a minipump) infusion for 2 weeks to induce hypertension. Endothelium-dependent relaxation (ED) was examined by organ bath, hematoxylin and staining and Masson-Trichrome staining were used to evaluate aorta and cardiac hypertrophy and fibrosis. Results: Ang II infusion significantly increased systolic blood pressure (SBP), cardiac hypertrophy and fibrosis, and impaired EDR accompanied by increased macrophage infiltration in the heart. Treatment with LEC significantly lowered Ang II-induced cardiac hypertrophy and fibrosis and cardiac macrophage infiltration, and improved EDR with a mild reduction in SBP. Ang II increased the expression of inflammatory cytokines tumor necross factor alpha and interleukin 1 beta and profibrotic factors transforming growth factor beta 1 and fibronectin in the heart, with was reduced by LEC treatment. Treatment with LEC prevented Ang II-induced the phosphorphorylation of ERK1/2 and c-Jun-N-terminal kinase. Conclusions: Our study suggests that cardiac macrophage may be critical for hypertensive cardiac hypertrophy and remodeling, the underlying mechanisms may involve initial heart inflammation and the activation of hypertrophic MAPKs pathway.

Abstract 265: Systemic Inflammation and Cardiac Macrophage Infiltration Accompany Stretch-induced Myocardial Injury in Swine Subjected to Transient Pressure Overload

Objective: Transient pressure overload (TPO) elicits stretch-induced myocyte injury in the absence of ischemia or infarction, but the extent to which this is associated with inflammation is unclear. The present study was designed to assess immune activation after TPO in swine and compare the inflammatory cytokine profile to that observed after myocardial infarction (MI). Methods: Swine received a brief infusion of phenylephrine (18 mg/hr iv; 30-60 min) to induce TPO and were studied for 1 hr (n=5), 3 hr (n=6), or 24 hr (n=6) before analysis of myocardial macrophage gene expression (CD68) via qPCR. Serial blood sampling was performed to assess neutrophil and monocyte counts as well as circulating cardiac troponin I (cTnI), IL-6, TNF-α, and CRP. Cytokine levels were compared to those from a separate group (n=5) subjected to a 60 min LAD occlusion to produce MI. Results: TPO increased circulating cTnI (from 20±5 ng/L to 340±58 ng/L at 3 hr and 1520±616 ng/L at 24 hr; p<0.01) without evidence of infarction. Compared with controls (n=6), swine subjected to TPO exhibited increased CD68 gene expression at 1, 3, and 24 hr ( A ). This was accompanied by a rise in peripheral blood neutrophils and monocytes ( B ) as well as an elevation in circulating IL-6, TNF-α, and CRP that was comparable to that observed after MI ( C ). Conclusion: Myocyte injury following TPO elicits mobilization of neutrophils and monocytes and a rise in circulating inflammatory cytokines comparable to that observed after MI. This is accompanied by an increase in cardiac macrophages and may be an important mechanism by which repetitive episodes of TPO lead to interstitial fibrosis and diastolic dysfunction without anatomic hypertrophy.

Macrophages represent the major pool of IL-7Rα expressing cells in patients with myocarditis

Myocarditis is characterized by infiltration and activation of cytokine as well as chemokine receptors frequently producing heart failure. Causes are often infections triggering inflammatory and immune responses but these initial lines of defense might be finally disastrous. To identify mediators we screened various receptors by confocal microscopy and identified cardiac interleukin-7 (IL-7) receptor-α (IL-7Rα) expressing cells in patients with myocarditis. IL-7Rα+ cells were analyzed by markers for leukocytes (CD45), B cells (CD19), T cells (CD3, CD4, CD8) and macrophages (CD68, CD163, CD206). Immune cells were hardly detected in controls. In patients with myocarditis main inflammatory populations consisted of macrophages and T cells. B cells were hardly present. 90% of CD68+ macrophages but less than 20% of CD3+ T cells were IL-7Rα+. This was surprising since T and B lymphocytes are generally regarded as the major IL-7Rα+ cells. Since IL-7 acts as a chemokine, the expression of its receptor might orchestrate cardiac macrophage infiltration. In contrast, consumption of IL-7 by IL-7Rα+ cardiac macrophages might potentially prevent a certain overshooting immune reaction and sepsis by reducing proliferation and survival of lymphocytes. Our data suggest a participation of IL-7Rα+ macrophages in the development of myocarditis and heart failure.

PHENOTYPIC HETEROGENEITY OF CARDIAC MACROPHAGES DURING WOUND HEALING FOLLOWING MYOCARDIAL INFARCTION: PERSPECTIVES IN CLINICAL RESEARCH

Purpose. Myocardial regeneration is one of the most ambitious goals in prevention of adverse cardiac remodeling. Macrophages play a key role in transition from inflammatory to regenerative phase during wound healing following myocardial infarction (MI). We have accumulated data on macrophage properties ex vivo and in cell culture. However, there is no clear information about phenotypic heterogeneity of cardiac macrophages in patients with MI. The purpose of the project was to assess cardiac macrophage infiltration during wound healing following myocardial infarction in clinical settings taking into consideration experimental knowledge.Material and Methods. The study included 41 patients with fatal MI type 1. In addition to routine analysis, macrophages infiltration was assessed by immunohistochemistry. We used CD68 as a marker for the cells of the macrophage lineage, while CD163, CD206, and stabilin-1 were considered as M2 macrophage biomarkers. Nine patients who died from noncardiovascular causes comprised the control group.Results. The intensity of cardiac macrophage infiltration was higher during the regenerative phase than during the inflammatory phase. Results of immunohistochemical analysis demonstrated the presence of phenotypic heterogeneity of cardiac macrophages in patients with MI. We noticed that numbers of CD68+, CD163+, CD206+, and stabilin-1+ macrophages depended on MI phase.Conclusion. Our study supports prospects for implementation of macrophage phenotyping in clinic practice. Improved understanding of phenotypic heterogeneity might become the basis of a method to predict adverse cardiac remodeling and the first step in developing myocardial regeneration target therapy.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

215 Cardiac macrophage infiltration during chronic kidney disease accelerates cardiovascular disease

The relationship between chronic kidney disease (CKD) and increasing rates of cardiovascular disease and mortality is complex, but is important due to the massive increased risk of cardiovascular events noted...

Sodium restriction modulates innate immunity and prevents cardiac remodeling in a rat model of metabolic syndrome

In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (<0.01% NaCl) for 8weeks. After 4weeks, rats were infused or not with angiotensin II (200ng·kg−1·min−1, sc) for 4weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry.Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hypertrophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet.Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling.

Green tea extract intake during lactation modified cardiac macrophage infiltration and AMP-activated protein kinase phosphorylation in weanling rats from undernourished mother during gestation and lactation.

Maternal dietary restriction is often associated with cardiovascular disease in offspring. The aim of this study was to investigate the effect of green tea extract (GTE) intake during lactation on macrophage infiltration, and activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and serine-threonine kinase Akt (Akt) in the hearts of weanlings exposed to maternal dietary protein restriction. Pregnant Wistar rats were fed control (C) or low-protein diets (LP) throughout gestation. Following delivery, the dams received a control or a GTE-containing control diet during lactation: control diet during gestation and lactation (CC), low-protein diet during gestation and lactation (LPC), low-protein diet during gestation and 0.12% GTE-containing low-protein diet during lactation (LPL), and low-protein diet during gestation and 0.24% GTE-containing low-protein diet during lactation (LPH). The female offspring were sacrificed at day 22. Biochemical parameters in the plasma, macrophage infiltration, degree of fibrosis and expression levels of AMPK and Akt were examined. The plasma insulin level increased in LPH compared with LPC. Percentage of the fibrotic areas and the number of macrophages in LPC were higher than those in CC. Conversely, the fibrotic areas and the macrophage number in LPH were smaller (21 and 56%, respectively) than those in LPC. The levels of phosphorylated AMPK in LPL and LPH, and Akt in LPH were greater than those in LPC. In conclusion, maternal protein restriction may induce macrophage infiltration and the decrease of insulin levels. However, GTE intake during lactation may suppress macrophage infiltration and restore insulin secretion function via upregulation of AMPK and insulin signaling in weanlings.

Metabolically-inactive glucagon-like peptide-1(9-36)amide confers selective protective actions against post-myocardial infarction remodelling.

BackgroundGlucagon-like peptide-1 (GLP-1) therapies are routinely used for glycaemic control in diabetes and their emerging cardiovascular actions have been a major recent research focus. In addition to GLP-1 receptor activation, the metabolically-inactive breakdown product, GLP-1(9–36)amide, also appears to exert notable cardiovascular effects, including protection against acute cardiac ischaemia. Here, we specifically studied the influence of GLP-1(9–36)amide on chronic post-myocardial infarction (MI) remodelling, which is a major driver of heart failure progression.MethodsAdult female C57BL/6 J mice were subjected to permanent coronary artery ligation or sham surgery prior to continuous infusion with GLP-1(9–36)amide or vehicle control for 4 weeks.ResultsInfarct size was similar between groups with no effect of GLP-1(9–36)amide on MI-induced cardiac hypertrophy, although modest reduction of in vitro phenylephrine-induced H9c2 cardiomyoblast hypertrophy was observed. Whilst echocardiographic systolic dysfunction post-MI remained unchanged, diastolic dysfunction (decreased mitral valve E/A ratio, increased E wave deceleration rate) was improved by GLP-1(9–36)amide treatment. This was associated with modulation of genes related to extracellular matrix turnover (MMP-2, MMP-9, TIMP-2), although interstitial fibrosis and pro-fibrotic gene expression were unaltered by GLP-1(9–36)amide. Cardiac macrophage infiltration was also reduced by GLP-1(9–36)amide together with pro-inflammatory cytokine expression (IL-1β, IL-6, MCP-1), whilst in vitro studies using RAW264.7 macrophages revealed global potentiation of basal pro-inflammatory and tissue protective cytokines (e.g. IL-1β, TNF-α, IL-10, Fizz1) in the presence of GLP-1(9–36)amide versus exendin-4.ConclusionsThese data suggest that GLP-1(9–36)amide confers selective protection against post-MI remodelling via preferential preservation of diastolic function, most likely due to modulation of infiltrating macrophages, indicating that this often overlooked GLP-1 breakdown product may exert significant actions in this setting which should be considered in the context of GLP-1 therapy in patients with cardiovascular disease.

DPP-4 inhibitor linagliptin ameliorates cardiovascular injury in salt-sensitive hypertensive rats independently of blood glucose and blood pressure.

BackgroundIt remains to be elucidated whether dipeptidylpeptidase-4 (DPP-4) inhibitor can ameliorate cardiovascular injury in salt-sensitive hypertension. The present study was undertaken to test our hypothesis that linagliptin, a DPP-4 inhibitor, administration initiated after onset of hypertension and cardiac hypertrophy can ameliorate cardiovascular injury in Dahl salt-sensitive hypertensive rats (DS rats).MethodsHigh-salt loaded DS rats with established hypertension and cardiac hypertrophy were divided into two groups, and were orally given (1) vehicle or (2) linagliptin (3 mg/kg/day) once a day for 4 weeks, and cardiovascular protective effects of linagliptin in DS rats were evaluated.ResultsLinagliptin did not significantly affect blood pressure and blood glucose levels in DS rats. Linagliptin significantly lessened cardiac hypertrophy in DS rats, as estimated by cardiac weight and echocardiographic parameters. Linagliptin significantly ameliorated cardiac fibrosis, cardiac macrophage infiltration, and coronary arterial remodeling in DS rats. Furthermore, linagliptin significantly mitigated the impairment of vascular function in DS rats, as shown by the improvement of acetylcholine-induced or sodium nitroprusside-induced vascular relaxation by linagliptin. These cardiovascular protective effects of linagliptin were associated with the attenuation of oxidative stress, NADPH oxidase subunits, p67phox and p22 phox, and angiotensin-converting enzyme (ACE).ConclusionsOur results provided the experimental evidence that linagliptin treatment initiated after the appearance of hypertension and cardiac hypertrophy protected against cardiovascular injury induced by salt-sensitive hypertension, independently of blood pressure and blood glucose. These beneficial effects of linagliptin seem to be attributed to the reduction of oxidative stress and ACE.

Glycemic control with empagliflozin, a novel selective SGLT2 inhibitor, ameliorates cardiovascular injury and cognitive dysfunction in obese and type 2 diabetic mice.

BackgroundThere has been uncertainty regarding the benefit of glycemic control with antidiabetic agents in prevention of diabetic macrovascular disease. Further development of novel antidiabetic agents is essential for overcoming the burden of diabetic macrovascular disease. The renal sodium glucose co-transporter 2 (SGLT2) inhibitor is a novel antihyperglycemic agent for treatment of type 2 diabetes. This work was performed to determine whether empagliflozin, a novel SGLT2 inhibitor, can ameliorate cardiovascular injury and cognitive decline in db/db mouse, a model of obesity and type 2 diabetes.Methods(1) Short-term experiment: The first experiment was performed to examine the effect of 7 days of empagliflozin treatment on urinary glucose excretion and urinary electrolyte excretion in db/db mice. (2) Long-term experiment: The second experiment was undertaken to examine the effect of 10 weeks of empagliflozin treatment on cardiovascular injury, vascular dysfunction, cognitive decline, and renal injury in db/db mice.Results(1) Short-term experiment: Empagliflozin administration significantly increased urinary glucose excretion, urine volume, and urinary sodium excretion in db/db mice on day 1, but did not increase these parameters from day 2. However, blood glucose levels in db/db mice were continuously decreased by empagliflozin throughout 7 days of the treatment. (2) Long-term experiment: Empagliflozin treatment caused sustained decrease in blood glucose in db/db mice throughout 10 weeks of the treatment and significantly slowed the progression of type 2 diabetes. Empagliflozin significantly ameliorated cardiac interstitial fibrosis, pericoronary arterial fibrosis, coronary arterial thickening, cardiac macrophage infiltration, and the impairment of vascular dilating function in db/db mice, and these beneficial effects of empagliflozin were associated with attenuation of oxidative stress in cardiovascular tissue of db/db mice. Furthermore, empagliflozin significantly prevented the impairment of cognitive function in db/db mice, which was associated with the attenuation of cerebral oxidative stress and the increase in cerebral brain-derived neurotrophic factor. Empagliflozin ameliorated albuminuria, and glomerular injury in db/db mice.ConclusionsGlycemic control with empagliflozin significantly ameliorated cardiovascular injury and remodeling, vascular dysfunction, and cognitive decline in obese and type 2 diabetic mice. Thus, empagliflozin seems to be potentially a promising therapeutic agent for diabetic macrovascular disease and cognitive decline.

GW24-e3848 The organ protective effect of direct renin inhibitor in a mouse model of type 2 diabetes

ObjectivesThe benefit of blocking the renin-angiotensin system (RAS) with conventional RAS blockers in cardiovascular diseases and nephropathy of patients with type 2 diabetes is now well established. Direct renin inhibitor...

Ezetimibe Ameliorates Cardiovascular Complications and Hepatic Steatosis in Obese and Type 2 Diabeticdb/dbMice

Type 2 diabetes plays a major role in the development of cardiovascular diseases. The present study was undertaken to investigate the effect of ezetimibe, a potent cholesterol absorption inhibitor, on cardiovascular injury of obese and type 2 diabetic db/db mice. Diabetic db/db mice fed a Western diet were given ezetimibe for 9 weeks, and the effects on cardiovascular injury and hepatic steatosis were examined. Ezetimibe treatment of db/db mice significantly improved vascular endothelial function, which was associated with the restoration of the decreased phospho-Akt and phospho-endothelial nitric-oxide synthase (eNOS). Moreover, ezetimibe also reduced vascular superoxide levels in db/db mice, accompanied by the attenuation of NADPH oxidase subunit gp91(phox) and Nox4 and the prevention of down-regulation of Cu/Zn-superoxide dismutase (SOD) and extracellular SOD. Thus, the improvement of vascular endothelial function by ezetimibe in diabetic mice seems to be attributed to the improvement of eNOS function and the attenuation of oxidative stress. Ezetimibe treatment also significantly attenuated cardiac interstitial fibrosis and coronary arterial thickening of diabetic mice and ameliorated cardiac macrophage infiltration. This improvement of cardiac injury was also related to the attenuation of NADPH oxidase-mediated oxidative stress. Furthermore, ezetimibe significantly prevented hepatic steatosis, inflammation, and oxidative stress in diabetic mice. Our work provides the first evidence that ezetimibe prevented cardiovascular injury and hepatic steatosis in diabetic mice. These beneficial effects were attributed to the attenuation of oxidative stress and inflammation and the improvement of eNOS function. Therefore, we propose that ezetimibe may be a promising therapeutic drug for obese and type 2 diabetes.

Potentiation by candesartan of protective effects of pioglitazone against type 2 diabetic cardiovascular and renal complications in obese mice

The efficacy of renin-angiotensin system (RAS) blockers on type 2 diabetes and its complications remains to be defined. This study was undertaken to test the hypothesis that candesartan may enhance the protective effects of pioglitazone against type 2 diabetes. We compared the effects of pioglitazone, candesartan, and their combination on cardiorenal and vascular injury, diabetes, and tissue oxidative stress in obese and type 2 diabetic db/db mice, and also examined the effects of tempol, a superoxide dismutase (SOD) mimetic, on db/db mice to define the role of oxidative stress. The addition of candesartan to pioglitazone significantly potentiated the suppressive effects of pioglitazone on cardiac macrophage infiltration and interstitial fibrosis, and glomerular macrophage infiltration and sclerosis in db/db mice. These benefits of the combination of pioglitazone and candesartan in db/db mice were attributed to additive attenuation of cardiorenal oxidative stress, through the attenuation of NADPH oxidase or the restoration of Cu/Zn-SOD and EC-SOD. The combination of these drugs reversed vascular endothelial dysfunction in db/db mice more than either monotherapy, by causing more phosphorylation of eNOS. Candesartan slightly augmented the improvement of glucose tolerance by pioglitazone in db/db mice, and this additive effect was mediated by more attenuation of oxidative stress. Our work demonstrated that candesartan significantly potentiated the protective effects of pioglitazone against cardiorenal and vascular injury, and diabetes in obese type 2 diabetic mice. Thus, the combination of pioglitazone with candesartan is potentially a promising therapeutic strategy for type 2 diabetes.

Abstract 4893: Selective Aldosterone Blocker Enhances Protective Effects of Calcium Channel Blocker (CCB) Against Salt-sensitive Hypertension-induced Cardiovascular Injury, Independently of Blood Pressure

Purpose : The effects of aldosterone blocker against hypertension (HTN)-induced cardiovascular injury are still unclear. We compared the protective effects of eplerenone (EPL), an aldosterone blocker and amlodipine (AML), a CCB on salt-sensitive HTN-induced cardiovascular injury, and examined these combined effects on cardiovascular protection. Methods : Dahl salt-sensitive rats fed on 8% NaCl containing diet were divided into vehicle, EPL (100mg/kg), AML (10mg/kg), and EPL combined with AML. Results : EPL monotherapy, without affecting blood pressure (BP) and plasma renin activity, significantly suppressed HTN-induced cardiac macrophage infiltration and interstitial fibrosis, coronary arterial remodeling, and improved HTN-induced vascular endothelial dysfunction, to a similar extent to strongly BP lowering AML. Beneficial effects of the combination therapy against HTN-induced cardiac inflammation, fibrosis, and vascular endothelial dysfunction were greater than AML monotherapy despite no lowering BP. Moreover, combination of EPL and AML markedly improved HTN-induced cardiac diastolic dysfunction compared with vehicle. These additive amelioration by the combination against hypertensive cardiac injury compared with AML monotherapy were associated with more attenuation of cardiac MCP-1, IL-6 and TGF- β 1 expressions, and more reduction of cardiac superoxide partially derived from NADPH oxidase. Furthermore, improvement of vascular endothelial dysfunction by the combination compared with AML monotherapy was attributed to not only more reduction of vascular NADPH oxidase-mediated superoxide but also more increase of NO by vascular endothelial NO synthase phosphorylation. It is interesting that the amelioration of endothelial fucntion by EPL was associated with the attenuation of HTN-induced vascular apoptosis signal-regulating kinase 1 (ASK1) activation. Actually, we found that vascular ASK1 deficiency markedly reduced vascular superoxide, and improved vascular endothelial dysfunction caused by aldosterone/salt-induced HTN in ASK1 deficient mice. Conclusions : Without lowering BP, aldosterone blocker enhanced protective effects of CCB against salt sensitive HTN-induced cardiovascular injury.

Critical Role of Apoptosis Signal-Regulating Kinase 1 in Aldosterone/Salt-Induced Cardiac Inflammation and Fibrosis

The molecular mechanism underlying aldosterone/salt-induced cardiovascular injury remains to be defined. This work was undertaken to determine the role of apoptosis signal-regulating kinase 1 (ASK1) in the mechanism underlying aldosterone-induced cardiac injury in vivo. We compared the in vivo effects of 4 weeks of aldosterone/salt treatment on wild-type and ASK1-deficient mice. Aldosterone infusion plus high salt intake in wild-type mice significantly increased blood pressure and urinary albumin excretion and decreased plasma potassium concentrations, and these effects of aldosterone/salt were not affected by ASK1 deficiency. Thus, ASK1 seems to play a minor role in aldosterone-induced hypertension and renal injury. ASK1 deficiency also failed to affect aldosterone-induced cardiac hypertrophy. However, ASK1 deficiency markedly ameliorated aldosterone-induced cardiac injury, eg, the enhancement of cardiac macrophage infiltration, monocyte chemotactic protein 1 expression, interstitial fibrosis, perivascular fibrosis, and transforming growth factor-beta1 and collagen type I expressions. Thus, ASK1 participates in aldosterone-induced cardiac inflammation and fibrosis. Furthermore, the enhancement of NADPH oxidase-mediated cardiac oxidative stress caused by aldosterone infusion was markedly lessened by ASK1 deficiency, which was associated with the significant amelioration by ASK1 deficiency of aldosterone-induced cardiac Nox2 upregulation. Furthermore, aldosterone/salt treatment significantly enhanced cardiac expression of the angiotensin-converting enzyme and angiotensin II type 1 receptor in wild-type mice, whereas the enhancement of these proteins by aldosterone/salt was abolished by ASK1 deficiency. Our results demonstrate that ASK1 is implicated in aldosterone/salt-induced cardiac inflammation and fibrosis through the enhancement of NADPH oxidase-mediated oxidative stress and the upregulation of the cardiac renin-angiotensin system.

Differential roles of cardiomyocyte and macrophage peroxisome proliferator-activated receptor gamma in cardiac fibrosis.

OBJECTIVE—Cardiac fibrosis is an important component of diabetic cardiomyopathy. Peroxisome proliferator–activated receptor γ (PPARγ) ligands repress proinflammatory gene expression, including that of osteopontin, a known contributor to the development of myocardial fibrosis. We thus investigated the hypothesis that PPARγ ligands could attenuate cardiac fibrosis.RESEARCH DESIGN AND METHODS—Wild-type cardiomyocyte- and macrophage-specific PPARγ−/− mice were infused with angiotensin II (AngII) to promote cardiac fibrosis and treated with the PPARγ ligand pioglitazone to determine the roles of cardiomyocyte and macrophage PPARγ in cardiac fibrosis.RESULTS—Cardiomyocyte-specific PPARγ−/− mice (cPPARγ−/−) developed spontaneous cardiac hypertrophy with increased ventricular osteopontin expression and macrophage content, which were exacerbated by AngII infusion. Pioglitazone attenuated AngII-induced fibrosis, macrophage accumulation, and osteopontin expression in both wild-type and cPPARγ−/− mice but induced hypertrophy in a PPARγ-dependent manner. We pursued two mechanisms to explain the antifibrotic cardiomyocyte-PPARγ–independent effects of pioglitazone: increased adiponectin expression and attenuation of proinflammatory macrophage activity. Adenovirus-expressed adiponectin had no effect on cardiac fibrosis and the PPARγ ligand pioglitazone did not attenuate AngII-induced cardiac fibrosis, osteopontin expression, or macrophage accumulation in monocyte-specific PPARγ−/− mice.CONCLUSIONS—We arrived at the following conclusions: 1) both cardiomyocyte-specific PPARγ deficiency and activation promote cardiac hypertrophy, 2) both cardiomyocyte and monocyte PPARγ regulate cardiac macrophage infiltration, 3) inflammation is a key mediator of AngII-induced cardiac fibrosis, 4) macrophage PPARγ activation prevents myocardial macrophage accumulation, and 5) PPARγ ligands attenuate AngII-induced cardiac fibrosis by inhibiting myocardial macrophage infiltration. These observations have important implications for potential interventions to prevent cardiac fibrosis.